JP2013061230A - Marker for determining boron satisfaction degree of plant and boron satisfaction degree determination method employing the same - Google Patents

Abstract

PROBLEM TO BE SOLVED: To develop and provide a boron satisfaction degree determination marker reflecting a boron satisfied/lacked state in a plant without being influenced by any environmental factor or the like, and to provide a method for determining the boron satisfied/lacked state in the plant by using the boron satisfaction degree determination marker.SOLUTION: A metabolic product which quantitatively varies depending on a boron satisfaction degree in a plant is used as a boron satisfaction degree determination marker. On the basis of the storage amount of the marker in a plant body, the boron satisfaction degree in the plant is determined accurately.

Description

  The present invention relates to a marker for determining a boron sufficiency state in a plant and a boron sufficiency determination method using the marker.

  As plant nutrients, nitrogen, phosphorus and potassium, which are called the three major nutrients, are best known, but in addition, many trace elements are known as nutrients essential for plant growth. One such trace element is boron.

  Boron is considered to be an element related to the formation of lignin and pectin and sugar transfer in plants, and although the required amount is small, its deficiency is known to cause abnormalities in plant meristems and formation layers. Yes. Therefore, for the normal growth of plants centering on the root tip and shoot apex, it is extremely important to provide boron appropriately and appropriately when necessary.

  In heavy rain regions such as Southeast Asia, boron in the soil flows out due to rainfall, and plant growth failure due to lack of boron is a major problem in agriculture and forestry. In general, it is known that when boron is deficient or deficient, various symptoms such as shoots die, roots become short, tree height is low, and the number of leaves is small. Therefore, it is necessary to take measures such as fertilizing boron on the land where such external symptoms occur in plants.

  However, in this region, plant growth defects are also caused by various factors other than boron, so it is difficult to determine that the cause is deficiency of boron only from the external symptoms of the plant. Therefore, if there is a technique that can accurately determine the sufficiency of boron in the plant, it becomes possible to perform appropriate boron fertilization according to the result, and the growth of the plant can be further promoted.

  Conventionally, boron sufficiency management in the field of agriculture and forestry has been performed by a method of measuring the amount of boron in soil (Non-patent Document 1). However, since the intake of various nutrients including boron is generally mediated by moisture in the soil, the amount of intake is greatly influenced by the amount of moisture in the soil. Therefore, this method is a method of measuring the boron content in an indirect field of soil, and does not measure boron that is substantially available to plants. Therefore, it cannot be said that the plant growing in the soil accurately reflects whether boron is satisfied or deficient, and it is difficult to say that it is a universally applicable method.

  As described above, no method has been known that can accurately and objectively determine the boron sufficiency of plants.

Japan Soil Association Foundation Soil, water quality and plant analysis method for soil function monitoring survey

  An object of the present invention is to develop and provide a marker for determining boron sufficiency that reflects the state of boron sufficiency and deficiency in plants without being influenced by environmental factors.

  Moreover, it is providing the method of determining the sufficiency and deficiency state of the boron in a plant using the said marker for boron sufficiency degree determination.

  In order to solve the above-mentioned problems, the present inventors have focused on plant metabolites and found metabolites that change quantitatively depending on the boron sufficiency in plants. In addition, based on the accumulated amount of the metabolite in the plant body, it has been clarified that the sufficient or insufficient state of boron in the plant can be accurately determined. This invention is based on the said knowledge, ie, provides the following.

（２）前記植物がユーカリプタス属である、（１）に記載のマーカー。
（３）植物体におけるホウ素の充足度を判定する方法であって、被検植物の全部又は一部から代謝産物を含む抽出物を抽出する抽出工程、前記抽出工程で得られた抽出物中に含まれる（１）に記載の少なくとも一のホウ素充足度判定用マーカーの蓄積量を測定する測定工程、前記測定工程で得られた蓄積量に基づいて、前記被検植物におけるホウ素の充足度を判定する判定工程を含む前記方法。
（４）前記判定工程において、ホウ素の充足度を、被検植物とホウ素欠乏状態の対照植物のそれぞれから得られるホウ素充足度判定用マーカーの蓄積量を比較したときに、被検植物における蓄積量が対照植物における蓄積量以上あるときには、その被検植物におけるホウ素が不足状態にあると判定する、（３）に記載の判定方法。
（５）被検植物がユーカリプタス属の種又はその雑種である、（３）又は（４）に記載の判定方法。
（６）（３）〜（５）のいずれかに記載の判定方法を用いて、その判定結果に基づいて被検植物を植栽する土地へのホウ素の施肥を決定するホウ素施肥方法。
（７）判定結果でホウ素が不足状態にあると判定された場合には、その被検植物へのホウ素施肥量を判定直前の量よりも増やす、（６）に記載のホウ素施肥方法。 (1) A metabolite of a plant, and in high-performance liquid chromatograph tandem mass spectrometry, when a temporal continuous concentration gradient of acetonitrile in liquid chromatography is formed from 0 min 3% to 6.3 min 70%, the following Boron of any one of the plants indicated by marker Nos. 1 to 5 which are separated by the retention times shown in Table 1 and whose mass-to-charge ratio of precursor ions and product ions in mass spectrometry is specified by the values shown in Table 1 below. Satisfaction degree marker.

(2) The marker according to (1), wherein the plant belongs to the genus Eucalyptus.
(3) A method for determining the degree of boron sufficiency in a plant body, wherein an extraction step for extracting an extract containing a metabolite from all or a part of a test plant, in the extract obtained in the extraction step A measurement step of measuring the amount of accumulation of at least one boron sufficiency determination marker described in (1), and determining the degree of boron sufficiency in the test plant based on the amount of accumulation obtained in the measurement step The method comprising the step of determining.
(4) In the determination step, when the accumulation level of the boron sufficiency determination marker obtained from each of the test plant and the boron-deficient control plant is compared with the boron sufficiency, the accumulation amount in the test plant (2) The determination method according to (3), wherein it is determined that boron in the test plant is in a deficient state when there is an accumulation amount in the control plant.
(5) The determination method according to (3) or (4), wherein the test plant is a species of the genus Eucalyptus or a hybrid thereof.
(6) A boron fertilization method that uses the determination method according to any one of (3) to (5) to determine fertilization of boron to the land on which the test plant is planted based on the determination result.
(7) The boron fertilization method according to (6), wherein when it is determined that the boron is in a deficient state based on the determination result, the amount of boron fertilization to the test plant is increased from the amount immediately before the determination.

  According to the marker for determining the boron sufficiency level of a plant of the present invention, it is possible to provide a marker that can accurately determine the boron sufficiency state of a plant without being influenced by other factors.

  According to the method for determining boron sufficiency of a plant of the present invention, by using the marker for determining boron sufficiency of a plant of the present invention, the boron sufficiency state in a test plant can be accurately determined without being influenced by other factors. can do.

  According to the boron fertilization determining method of the present invention, it becomes possible to fertilize the plant subjected to the inspection with an appropriate amount from the result obtained by the boron sufficiency determination method of the present invention.

It is the figure which showed the tree height of the Eucalyptus genus hybrid of the 1st year planting in a boron deficient area and a boron sufficient area (camaldrensis x eurofila). It can be seen that in the boron-deficient area, plant growth is significantly inhibited by boron deficiency.

1. Boron Satisfaction Determining Marker A first embodiment of the present invention is a plant boron sufficiency determining marker (hereinafter, simply referred to as “marker” in the present specification). The marker of the present invention is a plant-derived metabolite that changes quantitatively depending on the degree of boron filling in the plant. Specifically, it refers to substances indicated by marker Nos. 1 to 5 in Table 1 above.

  As used herein, “plant” refers to a moss plant, a fern plant, and a seed plant. Preferably it is a seed plant. In the case of a seed plant, a gymnosperm or angiosperm, a herb or a tree is not questioned. Preferred are angiosperms, more preferred are Myrtaceae plants, and still more preferred are plants of the genus Eucalyptus. For example, Eucalyptus Camaldrensis, Eucalyptus Deglupta, Eucalyptus Grandis, Eucalyptus Eurofila, Eucalyptus Perita, Eucalyptus Globras, Eucalyptus Brassiana, Eucalyptus Teleticontis or their hybrids, such as Eucalyptus Camaldrens Hybrids of cis and eucalyptus deglupta (hereinafter referred to as “camaldrensis x degrupta”), hybrids of eucalyptus camaldrensis and eucalyptus eurofila (hereinafter referred to as “camaldrensis x eurofila”), eucalyptus Examples include hybrids of Perita and Eucalyptus brassana, or hybrids of Eucalyptus perita and Eucalyptus camaldrensis.

  In the present specification, “the degree of sufficiency of boron in a plant” refers to the degree of whether or not boron has reached a necessary amount for the growth of the plant. For example, if the degree of boron sufficiency in a plant is low, it means that the amount of boron in the plant does not reach the amount necessary for growth, that is, the plant is in a deficient or deficient state of boron. . Further, when the degree of boron sufficient in a plant is high, it means that the amount of boron in the plant has reached the amount necessary for growth, that is, the plant is in a sufficient state of boron. In the present specification, a criterion for determining whether the degree of sufficiency of boron is high or low is determined based on the accumulated amount of the marker of the present invention. Since this will be described in detail in the determination step of the boron sufficiency determination method described later, the description thereof is omitted here.

  As used herein, the term “metabolite” refers to all substances produced in plants by catabolic metabolism represented by respiration or anabolic metabolism represented by photosynthesis. Examples include proteins (including enzymes) and low molecular weight compounds (including plant hormones, polyphenols, sugars, amino acids, and nucleotides). Water solubility and fat solubility do not matter.

  Among the above metabolites, the marker for determining the boron sufficiency according to the present invention is a high-performance liquid chromatograph tandem mass spectrometry, wherein the time continuous gradient of acetonitrile in liquid chromatography is from 0 min 3% to 6.3 min 70%. It is a substance indicated by marker Nos. 1 to 5 that is separated by the retention time shown in Table 1 when formed and the mass-to-charge ratio of precursor ions and product ions in mass spectrometry is specified by the values shown in Table 1. . More specifically, the substance is specified under the measurement conditions of Example 2 described later.

  “High-performance liquid chromatograph tandem mass spectrometry” (hereinafter abbreviated as “LC-MS / MS analysis”) is liquid chromatography (hereinafter abbreviated as “LC”). (MS: Mass Spectrometry) using a device in which two mass spectrometers are connected in series (Kachlickiet al) (Kachlickiet al). J Mass Spectrom. (2008) 43 (5): 572-586). In the LC-MS / MS system, a sample separated by LC at a specified retention time is ionized into precursor ions by the first MS (Q1) that can pass only ions having a specific mass-to-charge ratio, and then Q2 Metabolites are decomposed by high voltage and product ions are generated. In the subsequent second MS (Q3), only product ions having a specific mass-to-charge ratio pass and are detected by the detector. Therefore, even for metabolites whose chemical structures have not been identified, the sample separation conditions in LC and the mass-to-charge ratio of ions that can pass before and after decomposition are set in advance in the sample. The metabolite contained in can be identified.

  In the present invention, “a continuous concentration gradient of acetonitrile” refers to a continuous concentration gradient of acetonitrile, which is an LC solvent, within a predetermined time. Specifically, it refers to a concentration gradient in which the concentration at 0 minutes is 3%, the concentration at 6.3 minutes is 70%, and the concentration with respect to time in the meantime forms a continuous gradient.

  As described above, each boron sufficiency determination marker according to the present invention uses LC-MS / MS analysis to distribute a sample in a column together with acetonitrile that forms the temporal continuous concentration gradient. When the metabolites separated at the respective retention times described above are analyzed with a mass spectrometer, the precursor ions and the product ions are identified as substances having the mass-to-charge ratio shown in Table 1, the presence thereof is confirmed, and further quantification is performed. can do.

  In addition, as for the marker of this embodiment shown by marker No. 1-5 of Table 1, as shown in Example 1 mentioned later, all accumulate | store in the plant body with the decrease in the amount of boron, ie, Boron deficiency or deficiency is a metabolite having properties that increase the amount of expression, synthesis, or secretion or release in the test plant.

2. Boron sufficiency determination method The second embodiment of the present invention is a boron sufficiency determination method in plants. The method of the present invention includes an extraction step, a measurement step, and a determination step as essential steps. Hereinafter, each step will be specifically described.

2-1. Extraction Step The “extraction step” is a step of extracting an extract containing a metabolite from all or part of the test plant.

  In the present specification, the “test plant” refers to a plant that is used in the method for determining the boron sufficiency of the present invention in order to determine the sufficiency of boron, and is a plant grown under any environment.

  In this specification, “the whole test plant” means all parts constituting the plant body of the test plant. The “part of the test plant” means an organ (for example, root, stem, leaf, flower, spore or seed) constituting the plant of the test plant, or a form constituting the organ. It refers to a tissue that is a group of cells that have been differentiated objectively and / or functionally, or cells that constitute the tissue. The part may use any part of the test plant, but is preferably a leaf part. This includes the marker for determining the boron sufficiency according to the first embodiment because it is easily available, has a relatively small load on the test plant, and contains the most metabolites among the plants. This is because there is a high possibility that

  In the present specification, the “extract” refers to a plant extract including a plurality of metabolites, usually a plant extract. As described above, since the metabolite of the present invention may be water-soluble or fat-soluble, the solvent used for extraction of the extract may be an aqueous solution or an organic solvent (for example, lower alcohol, ether, chloroform, ethyl acetate, Xylene, etc.).

  The method for extracting the extract from the test plant is not particularly limited as long as it is a method capable of extracting a metabolite from the test plant. For example, when obtaining a water-soluble metabolite, the plant body is crushed as necessary, then watered, soaked and / or squeezed for a predetermined period, and then filtered to collect the filtrate or centrifuged. What is necessary is just to collect | recover subsequent supernatants. In addition, when obtaining a fat-soluble metabolite, the plant body is pulverized as necessary, then immersed in an organic solvent such as methanol for a predetermined period, and then filtered to collect the filtrate or centrifuged. The supernatant may be collected. For the details of these extraction methods, techniques known in the art can be used. For example, a method described in a literature name (Iijima et al., The Plant Journal (2008) 54,949-962, Suzuki et al., Phytochemistry (2008) 69, 99-111) may be referred to.

  In the boron sufficiency determination method of the present invention, in the determination step described later, when determining the sufficiency of boron by comparing the amount of accumulated marker obtained from each of the test plant and the control plant, It is desirable to extract the extract from the control plant as well as the test plant. In this case, it is assumed that the control plant is the same species or the same hybrid as the test plant and is in a deficient state lacking boron. The marker of the present invention includes other conditions other than the amount of boron, such as growth weather conditions (for example, temperature, sunshine duration, humidity), soil conditions, temporal conditions (for example, growth period, growth season) and the plant. These conditions do not necessarily have to be the same for the test and control plants because they are not affected or hardly affected by the health condition, but for more accurate results, boron It is preferred that each condition other than the amount is the same as much as possible. Moreover, when extracting an extract from a part of individual | organism | solid, it is preferable that the part uses the same part between a test plant and a control plant. For example, when the extract is extracted from the leaves of the test plant, the extract of the control plant is preferably extracted from the leaves as well.

2-2. Measurement step The “measurement step” is a step of measuring the amount of accumulated marker contained in the extract obtained in the extraction step.

  The marker to be measured in this step is at least one of the five markers described in the first embodiment, and any one may be selected arbitrarily. The use of a plurality of markers of 2 or more and 5 or less is also preferable for improving the reliability of the determination result in the determination method of the present invention.

  In the present specification, the “accumulated amount” is the amount of a specific marker in the extract obtained in the extraction step. This amount may be a relative amount such as concentration, ionic strength, absorbance or fluorescence intensity, or may be an absolute amount such as the weight or volume of a marker included in a given amount of extract. .

  The method for measuring the amount of accumulated marker contained in the extract is not particularly limited as long as it can detect and quantify a specific metabolite from the extract. For example, detection and quantification can be performed using mass spectrometry, a method applying an antigen-antibody reaction, electrophoresis and the like.

  Mass spectrometry includes high performance liquid chromatography mass spectrometry (LC-MS), high performance liquid chromatography tandem mass spectrometry (LC-MS / MS), gas chromatography mass spectrometry (GC-MS), and gas chromatography tandem mass spectrometry. Methods (GC-MS / MS), capillary electrophoresis mass spectrometry (CE-MS) and ICP mass spectrometry (ICP-MS).

  The antigen-antibody reaction is applied using an antibody that specifically recognizes each marker, ELISA (Enzyme-Linked ImmunoSorbent Assay) method, surface plasmon resonance (SPR) method, or quartz crystal microbalance (QCM) The law can be used.

  When the metabolite to be analyzed is a protein, for example, two-dimensional electrophoresis can be used as the electrophoresis.

  Any of the above analysis methods are known in the art, and may be performed according to these methods. For example, literature names (Yoko Iijima et al., The Plant Journal (2008) 54,949-962, Masami Hirai et al. Proc Natl Acad Sci USA (2004) 101 (27) 10205-10210, Shigeru Sato et al., The Plant Journal (2004) 40 (1) 151-163, Motoyuki Shimizu et al., Proteomics (2005) 5,3919-3931).

  Although the chemical structure of the marker described in the first embodiment has not yet been identified, since it can be identified by LC-MS / MS analysis as described in the first embodiment, the accumulated amount contained in the extract is It can be measured at least using LC-MS / MS analysis. For example, in order to measure the accumulated amount of marker No. 1 contained in the extract, acetonitrile in LC is 3% at 0 minutes, 70% at 6.3 minutes, The substance that flowed into the column with the extract to form a continuous concentration gradient with respect to time and separated in 2.37 minutes, the mass-to-charge ratio of precursor ions in Q1 to 499.2, and the mass of product ions in Q3 By passing through a tandem mass spectrometer set to a charge ratio of 499.2, if marker No. 1 is present in the extract, it can be specifically detected and quantified by the detector.

2-3. Determination Step The “determination step” is a step of determining whether the boron sufficiency in the test plant is high or low based on the marker accumulation amount obtained in the measurement step. “Based on the amount of accumulated marker” means that the amount of accumulated marker is used to determine the degree of boron sufficiency.

  The criterion for determining the degree of boron satisfaction is not particularly limited as long as it uses the accumulated amount of the marker. For example, a value set when selecting a marker or an obtained value may be used as a reference value as a reference value, or an accumulated amount of marker in a control plant may be used as a reference value.

  Here, the “reference value” is an ionic strength value in the LC-MS analysis obtained or used when selecting the marker shown in Table 1 as a marker for determining the degree of boron satisfaction.

  The marker of the present invention was obtained by hydroponically cultivating the same plant species as the test plant in the boron-satisfied and boron-deficient zones as described in Example 1 below, and was obtained from each individual in both zones. When comparing the accumulated amount of metabolites, as shown in Table 2 below, the selected amount of metabolites with an ionic strength that is less than 1 / 1.5 times the ionic strength of the accumulated amount in the boron-sufficient zone is there. That is, in Tables 1 and 2, the markers indicated by marker Nos. 1 to 5 are metabolites having a property that the accumulated amount in the plant body increases as the boron amount decreases. As shown in Table 2, the accumulated amounts of these markers in boron-deficient individuals are ionic strength, 0.50 ± 0.19 for marker No.1, 0.78 ± 0.34 for marker No.2, 1.96 for marker No.3. Since it was ± 0.84, 3.89 ± 1.14 for marker No. 4 and 5.47 ± 1.32 for marker No. 5, a value corresponding to 1 / 1.5 times the ionic strength of each, specifically marker No. 1 The reference value can be 0.33 ± 0.19, 0.52 ± 0.34 for marker No. 2, 1.31 ± 0.84 for marker No. 3, 2.59 ± 1.14 for marker No. 4, and 3.66 ± 1.32 for marker No. 5.

  Therefore, in marker No. 1-5, as the determination method, when the accumulated amount obtained in the test plant is ionic strength, which is not more than the reference value, the degree of boron sufficiency in the test plant is It can be determined that the amount is high, and conversely, when the reference value is exceeded, it can be determined that the boron sufficiency in the test plant is low.

  In addition, the accumulated amount of markers No. 1 to 5 in the boron-satisfied individual was ionic strength, 0.00 ± 0.00, 0.02 ± 0.02, 0.01 ± 0.01, 0.01 ± 0.01, and 0.07 ± 0.24, respectively. This value may be a stricter reference value. In this case, if the accumulated amount of the marker obtained in the test plant is not more than the reference value in terms of ionic strength, it is determined that the degree of boron sufficiency in the test plant is high, and conversely the reference When exceeding a value, it can also be determined that the boron sufficiency in the test plant is low.

  When using the above reference values in the determination process, the accumulated amount of markers analyzed by LC-MS analysis may cause slight errors between samples. It is desirable to standardize by dividing by the accumulated amount of the substance (eg (-) Epicatechin).

  Further, when the accumulated amount of the marker in the control plant is used as a criterion, the accumulated amount in the extract of the test plant is compared with the accumulated amount in the extract of the control plant for the marker of the present invention, and the result What is necessary is just to determine the sufficiency of boron in a test plant based on this. The term “control plant” as used herein refers to a plant that is the same species or hybrid as the test plant and has a growth state of the same level, and is a boron-satisfied individual to which boron is added in a minimum amount necessary for growth, or boron. And boron deficient individuals lacking. However, as described above, all of the five markers of the present invention are metabolites that are hardly accumulated in boron-satisfied individuals. Therefore, the control individual is preferably a boron-deficient individual.

  When the accumulated amount of the marker in the control plant is used as a criterion, it is preferable that the extraction step and the measurement step are performed under the same conditions at the same time in the control plant and the test plant. Alternatively, the extraction step and the measurement step are performed on the control plant under various conditions, and the accumulated amount of the obtained marker is stored in a database in advance, and in this step, various conditions other than boron are the test plant. The accumulated amount of the control plant marker that matches as much as possible may be selected and used.

  When determining the boron sufficiency of the test plant using the accumulated amount of the marker in the control plant as a determination criterion, the specific determination method is not limited. For example, if the control plant is a boron-deficient individual lacking boron or a boron-deficient individual having a boron content below the minimum required for plant growth, the accumulated amount of the marker in the test plant and the control plant is statistically determined. Whether or not a significant quantitative difference is observed is verified, and if there is a significant difference, it can be determined that boron in the test plant is in a sufficient state. “Statistically significant” means that there is a significant difference when the difference in the amount of accumulated marker is statistically processed. The test method for statistical processing is not particularly limited as long as a known test method capable of determining the presence or absence of significance is appropriately used. For example, Student's t test or multiple comparison test can be used. Specifically, for example, the risk rate (significance level) is less than 5%, 1%, or 0.1%.

  Since all of the markers of the present invention are inversely proportional to the amount of boron in the plant, as a specific example of a statistically significant quantitative difference, the amount of accumulated marker in the control plant is ionic strength, boron deficient state In the control plant, the ionic strength is 1 / 1.5 times or less, preferably 1/2 times or less, more preferably 1 / 2.5 times or less.

  According to this method, the boron nutritional state in the test plant can be accurately determined without being influenced by other factors.

  According to this method, the amount of boron that can be substantially used by the plant in the planting site where the plant is planted can be determined from the boron sufficiency of the plant.

  According to this method, by using a plant that naturally grows on a specific land, it is possible to determine the boron sufficiency of the land before using it as a planting place by determining the sufficiency of the boron.

3. Boron fertilization method The third embodiment of the present invention is a boron fertilization method. This method is a method of determining boron fertilization to the land where the test plant is planted based on the determination result using the boron sufficiency determination method of the second embodiment.

  The determination as to whether or not to fertilize boron may be made as appropriate based on the determination result of the boron sufficiency determination method of the second embodiment. For example, when it is determined that the boron satisfaction level of the test plant is in an insufficient state, fertilization is performed immediately before the boron to be fertilized on the land where the test plant is planted is used for the boron satisfaction level determination method. If the amount is increased or the boron is not fertilized, fertilization may be performed. In addition, when it is determined that the boron sufficiency level of the test plant is in a satisfactory state, fertilization is performed immediately before the amount of boron to be applied to the land where the test plant is planted is provided to the method for determining the boron sufficiency level. It is sufficient to maintain the amount as it was.

  The boron to be fertilized may be fertilized in a state known in the art. For example, it can be fertilized as boric acid or borate such as borax.

  According to this method, it is possible to determine whether and how much boron should be fertilized on the land where the test plant grows, and how much fertilization should be performed. It also makes it possible to avoid or eliminate the boron deficiency or deficiency state of the plant and maintain or improve the growth state of the plant. As a result, plant production and production efficiency can be increased.

<Example 1: Selection of a marker for determination of boron sufficiency>
(material)
24 hybrid clones of the genus Eucalyptus (camaldrensis x deglupta) that were hydroponically cultivated for about 3 months in two test plots (boron-fulfilled zone and boron-deficient zone) were used. As for the cultivation conditions in both test plots, in the boron sufficient plot, the liquid medium contains the minimum amount of boron (1.5 mg / L) necessary for growth, and in the boron deficient plot, the amount of boron in the liquid medium is zero. Except for this, everything is the same.

  In the boron-deficient clones, growth inhibition characteristic of boron deficiency was observed, such as shoot death, root shortening, few leaves, and low tree height.

(Method)
1. Leaf Sampling For each Eucalyptus individual in the above boron-sufficient and boron-deficient areas, leaves were cut out from the petiole portion with scissors. The main vein portion of the collected leaves was removed, and the rest was dried at 50 ° C. overnight.

2. Extraction of Extract Each individual of about 30 mg in dry weight was placed in a ceflock tube. A zirconia ball (Tosoh) having a diameter of 5 mm was added to the tube, and the leaf sample was pulverized with Tissue Lyser II (Qiagen) at 25 Hz for 30 seconds. Next, add 1 mL of extract (water: methanol: chloroform = 1: 2.5: 1 (v: v: v), 5 mg / mL (-) containing Epicatechin as an internal standard substance) to the tube, and add Tissue Lyser II. And extracted by shaking for 120 seconds at 25 Hz. The obtained extract was centrifuged at 12000 × g for 10 minutes, 0.4 mL of water was added to about 0.9 mL of the supernatant, and centrifuged at 12000 × g for 10 minutes, and the upper layer (aqueous methanol layer) and the lower layer (chloroform) Layer). Subsequently, the upper layer was filtered using Millex-LG (Millipore), and the filtrate was recovered.

3. LC-MS analysis 10 μL of the obtained filtrate was subjected to LC-MS analysis under the following conditions. About the specific analysis method, the instruction manual attached to each equipment used was followed.

3-1. Liquid Chromatography (LC) Conditions • Equipment used: UFLC (Shimadzu Corporation)
・ Eluent A: 0.1% formic acid (Wako) containing water resolve for water chromatography
・ Eluent B: Hypergrade resolvosolve for acetonitrile LC / MS with 0.1% formic acid (Wako)
・ Column: TSKgel ODS-100V 3.0x 750mm particle 3μm (Tosoh)
Guard column: TSKguardgel ODS-100V, particle 5μm (Tosoh)
・ Column temperature: 40 ℃
・ Flow rate: 0.5 mL / min
・ Time continuous gradient (time schedule gradient) B%
0 minutes: 3% to 10 minutes: 97%

3-2. MS conditions-Equipment used: 3200QTRAP
・ Mass range: 100-1000
・ Ionization method: ESI-Positive
-Scan mode: Enhanced Mass Scan
・ Scanning speed: 4000 amu / sec
・ Analysis software: Analyst 1.4.2

3-3. MSMS conditions-Equipment used: 3200QTRAP
・ Mass range: 50-1000
・ Ionization method: ESI-Positive
-Scan mode: Enhanced Product Ion Scan
・ Scanning speed: 4000 amu / sec
・ Analysis software: Analyst 1.4.2

3-4. Alignment conditions-Metabolite data alignment: Marker View ^TM 1.2 Software
・ Elution time tolerance: 1 minute ・ Mass tolerance: 25 ppm
・ Intensity threshold: 10,000

4). Selection of markers for determining boron sufficiency From the metabolites obtained in the LC-MS / MS analysis, select metabolites that show boron sufficiency in terms of accumulated amount using Volcano Plot of GeneSpring MS (Agilent Technologies). did. Specifically, when the accumulated amount of metabolites was compared between individuals in the boron-sufficient group and those in the boron-deficient group, the accumulated amount in the sufficiency-dwelling individual was more than 1.5 times that in the deficient-deficient individual in terms of ionic strength A product or a metabolite of 1 / 1.5 times or less was selected as a marker for determining the boron sufficiency.

  All metabolites are substances that vary in the plant depending on the amount of boron, but the former has a proportional relationship with the amount of boron, that is, the amount of accumulation in the plant increases as the amount of boron increases. On the contrary, the latter can be said to be a marker having an inversely proportional relationship with the amount of boron, in which the accumulated amount in the plant body decreases as the amount of boron increases.

(result)
As a result of selection, five metabolites (markers No. 1 to 5) shown in Tables 2 and 3 were obtained. Table 2 shows the results of comparing the amount of accumulated metabolites between boron-deficient individuals and boron-sufficient individuals, and Table 3 is for identifying each of the obtained markers by LC-MS / MS analysis. LC retention time, mass-to-charge ratio of precursor ion and product ion.

  As shown in this table, marker Nos. 1 to 5 are all accumulated in the deficient individual, but are rarely found in the sufficiency individual, and the accumulated amount in the sufficiency individual is the ionic strength in the deficient individual It became clear that it is a metabolite classified as less than 1 / 1.5 times that. That is, the metabolites of the markers Nos. 1 to 5 of the present invention were markers having a property that is inversely proportional to the boron content.

<Example 2: Determination of boron sufficiency of test plant using boron sufficiency determination method>
In a plantation site that is considered to be affected by complex environmental factors, the boron sufficiency determination method of the present invention is used for a eucalyptus tree species sample different from that in Example 1, and the actual boron sufficiency is accurately measured. It was verified whether the judgment was possible.

(material)
A 0.8 year-old Eucalyptus hybrid planted in two test sites (Test Site A and Test Site B) in Laos plantation was used as a sample. In these two test sites, a boron fertilization zone and a non-fertilization zone were set for each, and boron fertilized individuals and boron non-fertilized individuals were used from test zones A and B, respectively. In both test plots, boron-deficient fertilizer plots showed typical boron deficiency symptoms (such as dead shoots and small number of leaves).

(Method)
1. Leaf Sampling Each individual leaf in the boron fertilization section and boron non-fertilization section in both test sections was cut from the petiole portion with scissors. The main vein portion of the collected leaves was removed, and the rest was dried at 50 ° C. overnight.

2. Extraction Extraction The extraction was performed according to the method described in Example 1.

3. LC-MS / MS analysis 10 μL of the obtained filtrate was subjected to LC-MS / MS analysis under the following analysis conditions. About the specific analysis method, the instruction manual attached to each equipment used was followed.

3-1. Liquid Chromatography (LC) Conditions • Equipment used: UFLC (Shimadzu Corporation)
・ Eluent A: 0.1% formic acid (Wako) containing water resolve for water chromatography
・ Eluent B: Hypergrade resolvosolve for acetonitrile LC / MS with 0.1% formic acid (Wako)
・ Column: TSKgel ODS-100V 3.0x 750mm particle 3μm (Tosoh)
Guard column: TSKguardgel ODS-100V, particle 5μm (Tosoh)
・ Column temperature: 40 ℃
・ Flow rate: 1.0 ml / min
・ Time continuous gradient (time schedule gradient) B%
0 minutes: 3% to 6.3 minutes: 70%

3-2. MS conditions-Equipment used: 3200QTRAP
-Scan mode: Multiple Reaction Monitoring
・ Ionization method: ESI-Positive
・ Scanning speed: 4000 amu / sec
・ Analysis software: Analyst 1.4.2
・ Target Mass:
No.1: Q1: m / z: 499.2, Q3: m / z: 499.2 RT = 2.4
No.2: Q1: m / z: 497.2, Q3: m / z: 497.2 RT = 3.5
No.3: Q1: m / z: 475.1, Q3: m / z: 313.2 RT = 4.8
No.4: Q1: m / z: 461.2, Q3: m / z: 299.2 RT = 4.7
No.5: Q1: m / z: 673.6, Q3: m / z: 673.6 RT = 4.5
No.6: Q1: m / z: 291.2, Q3: m / z: 139.1 RT = 2.6
Here, No. 6 is an internal standard substance (-) Epicatechin.
Q1 and Q3 are mass-to-charge ratio data (m / z) of each marker, and RT is a retention time (min).

4). (Standardization of data)
In the case of a mass spectrometer, the amount of accumulated marker is slightly different for each analysis even if it is the same sample, so standardization is required. Here, the accumulation amount of various markers was standardized by dividing by the accumulation amount of the internal standard substance ((−) Epicatechin).

5. Discriminant analysis The metabolite data obtained from the boron fertilizer sample and non-fertilized sample at test site A is used as a training data set for discriminant analysis, that is, a base text sample, and the test site as the test sample Based on the profile of all the markers shown in Table 1, the B sample (total 52 specimens) was evaluated as being sufficient or insufficient for boron. The determination method was performed under the following conditions.

-Judgment algorithm: K nearest neighbor method In the case of a class classification that is given a training case with a class label: find k cases in order from the one closest to the case to be classified. A method of classifying the k cases into the class with the largest number.
・ Marker: 5 types of markers for determining boron sufficiency ・ Training data set: Obtained from boron fertilizer sample (boron-sufficient individuals) and boron-free fertilizer sample (boron-deficient individuals) set to test site A in Laos plantation Marker profiles and test sample data: Marker profiles obtained from each of 52 boron fertilization test samples (27 boron fertilization sample and 25 boron non-fertilization sample) set at test site B in Laos plantation

(result)
The results are shown in FIG.
When the boron sufficiency of the 27 samples in the boron fertilization section of the test site B was determined, a determination result that all the samples were in a satisfiable state in all the markers was obtained. Since the sample in the boron fertilized section is a boron-satisfied individual, a correct answer in which the determination result matches the actual condition is obtained by the determination method of the present invention.

  On the other hand, a discriminant analysis of the 25 samples without fertilization with boron revealed that 4 individuals were in a satisfactory state, but 21 samples had a lack of boron. Since the sample in the non-boron fertilized section is a boron-deficient individual, approximately 92.3% of correct answers were obtained by the determination method of the present invention.

  As described above, according to the boron sufficiency determination method using the marker of the present invention, a high determination exceeding 90% of the correct answer rate even for a plantation site sample considered to be affected by a complicated environmental factor. It was proved that the boron sufficiency of plants growing on the land can be judged with accuracy.

  Further, as described above, the plant species used in the experiment are different between Example 1 and Example 2. The marker of the present invention is a metabolite isolated from the plant species described in Example 1. However, in this example, it was revealed that the marker was also effective for different plant species, and the generality of the marker was proved. .

Claims (7)

A metabolite of a plant, and in high performance liquid chromatography tandem mass spectrometry,
When a time continuous gradient of acetonitrile in liquid chromatography was formed from 0 min 3% to 6.3 min 70%, it was separated with the retention times shown in Table 1 below, and precursor ions and product ions in mass spectrometry A marker for determining the boron sufficiency of any one of the plants indicated by marker Nos. 1 to 5 specified by the values shown in Table 1 below.

  The marker according to claim 1, wherein the plant belongs to the genus Eucalyptus. A method for determining the degree of boron fulfillment in a plant,
An extraction step for extracting an extract containing a metabolite from all or a part of the test plant;
A measurement step of measuring an accumulated amount of at least one boron sufficiency determination marker according to claim 1 contained in the extract obtained in the extraction step,
The said method including the determination process of determining the sufficiency of the boron in the said test plant based on the accumulation amount obtained at the said measurement process.   In the determination step, when the boron sufficiency is compared with the amount of accumulation of the boron sufficiency determination marker obtained from each of the test plant and the boron deficient control plant, the boron sufficiency determination in the test plant The determination method according to claim 3, wherein when the accumulated amount of the marker is greater than or equal to the accumulated amount in the control plant, it is determined that boron in the test plant is in a deficient state.   The determination method according to claim 3 or 4, wherein the test plant is a species of the genus Eucalyptus or a hybrid thereof.   The boron fertilization method which determines the fertilization of the boron to the land which plants a test plant based on the determination result using the determination method as described in any one of Claims 3-5.   The boron fertilization method of Claim 6 which increases the amount of boron fertilization to the to-be-tested plant from the amount just before determination, when it determines with the determination result showing that the boron is in a deficient state.

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